In Silico Analysis of PORD Mutations on the 3D Structure of P450 Oxidoreductase

Cytochrome P450 oxidoreductase (POR) is a membrane-bound flavoprotein that helps in transferring electrons from its NADPH domain to all cytochrome P450 (CYP450) enzymes. Mutations in the POR gene could severely affect the metabolism of steroid hormones and the development of skeletal muscles, a condition known as Cytochrome P450 oxidoreductase deficiency (PORD). PORD is associated with clinical presentations of disorders of sex development, Antley and Bixler’s syndrome (ABS), as well as an abnormal steroid hormone profile. We have performed an in silico analysis of POR 3D X-ray protein crystal structure to study the effects of reported mutations on the POR enzyme structure. A total of 32 missense mutations were identified, from 170 PORD patients, and mapped on the 3D crystal structure of the POR enzyme. In addition, five of the missense mutations (R457H, A287P, D210G, Y181D and Y607C) were further selected for an in-depth in silico analysis to correlate the observed changes in POR protein structure with the clinical phenotypes observed in PORD patients. Overall, missense mutations found in the binding sites of POR cofactors could lead to a severe form of PORD, emphasizing the importance of POR cofactor binding domains in transferring electrons to the CYP450 enzyme family.


Introduction
Cytochrome P450 oxidoreductase (POR), previously known as cytochrome c reductase, is an essential membrane-bound flavoprotein that transfers electrons to all the cytochrome P450 (CYP450) enzymes, thus allowing them to fulfill their actions effectively [1][2][3]. Besides providing electrons to all the microsomal P450 enzymes, POR also plays a crucial role in the process of metabolizing drugs, steroid hormone synthesis, and xenobiotics metabolism ( Figure 1) [1]. Furthermore, the importance of POR in transferring electrons is not just limited to microsomal CYP450; in fact, other enzymes such as heme oxygenase, cytochrome b5, and squalene monooxygenase, as well as 7-dehydrocholesterol reductase rely on POR to work efficiently ( Figure 1) [4]. The gene of POR in homo sapiens contains a total of 16 exons-1 non-coding exon and 15 protein-coding exons-which code for a membrane-bound protein containing 680 amino acids [1]. The POR gene, which is located on chromosome 7q11.23, is anchored towards the endoplasmic reticulum via a hydrophobic N-terminus domain that helps in the interaction with CYP450 enzymes [5].
In terms of its 3D structure, POR comprises three cofactor binding domains, which are the flavin adenine dinucleotide (FAD) binding domain, the flavin adenine mononucleotide (FMN) binding domain, and the NADPH binding domain (Figures 2 and 3) [6,7]. Moreover, the two flavin cofactors (FAD and FMN) are closely connected together via a flexible hinge region that can allow the movement of electrons by undergoing conformational changes, bringing FAD nearer to the domain of FMN (Figures 2 and 3) [5]. The initial step of transferring electrons within POR begins with NADPH being oxidized as it donates Figure 1. Role of cytochrome P450 oxidoreductase (POR). POR provides electrons to all microsomal P450 enzymes that play a crucial role in the process of metabolizing drugs, steroid hormone synthesis, and xenobiotic metabolism, as well as to other enzymes that also rely on POR to work efficiently, such as heme oxygenase, cytochrome b5, squalene monooxygenase as well as 7dehydrocholesterol reductase.  (POR). POR provides electrons to all microsomal P450 enzymes that play a crucial role in the process of metabolizing drugs, steroid hormone synthesis, and xenobiotic metabolism, as well as to other enzymes that also rely on POR to work efficiently, such as heme oxygenase, cytochrome b5, squalene monooxygenase as well as 7-dehydrocholesterol reductase.
Molecules 2022, 27, x FOR PEER REVIEW 2 of 14 via a flexible hinge region that can allow the movement of electrons by undergoing conformational changes, bringing FAD nearer to the domain of FMN (Figures 2 and 3) [5]. The initial step of transferring electrons within POR begins with NADPH being oxidized as it donates its hydride anion (H-) to FAD which will eventually get reduced, the H-ion will then be accepted by FMN, which will pass the electrons to the CYP450 enzyme, specifically the heme center. FAD will receive two electrons from NDAPH but will only transfer one electron to FMN at a time. Similarly, FMN will finally provide the electrons to CYP450 one at a time [4].

Figure 1.
Role of cytochrome P450 oxidoreductase (POR). POR provides electrons to all microsomal P450 enzymes that play a crucial role in the process of metabolizing drugs, steroid hormone synthesis, and xenobiotic metabolism, as well as to other enzymes that also rely on POR to work efficiently, such as heme oxygenase, cytochrome b5, squalene monooxygenase as well as 7dehydrocholesterol reductase.

POR Deficiency (PORD)
A number of gene mutations have been reported in the POR enzyme, resulting in the distortion of its 3D protein structure, ultimately causing POR to function abnormally. The mutation-driven changes in the 3D structure of the POR enzyme could lead to a medical condition known as cytochrome P450 oxidoreductase deficiency (PORD) (OMIM: 613571), which is an autosomal recessive genetic disorder and can also be categorized as a rare congenital adrenal hyperplasia (CAH) [3,8]. PORD has an impact on the regulation of steroid hormone production in the body; thus, this condition could affect the normal development of the skeletal system, the reproductive system, as well as metabolic pathways. In an early experiment, Peterson et al. (1985) [9] assessed a 46, XY, six-monthold infant characterized by having a female phenotype and abnormal genitals, with an unusual steroid profile, and it was assumed to be caused by a loss of function of both CYP17A1 and CYP21A2. This report led Miller et al. (1986) [10] to propose that there might be a defect in the POR enzyme, which provides electrons to both CYP17A1 and CYP21A2. In spite of that, the hypothesis was excluded as an embryonic lethality was discovered in POR knockout mice [11,12]. It was not until in 2004, when Flück et al. [3] confirmed that the first four individuals suffering from PORD have mutations in their POR gene. The author also described how three out of four patients developed abnormal genitals while the remaining 1 patient suffered from an abnormal steroid profile, a cystic ovary, and primary amenorrhea [3].
As PORD is a rare autosomal recessive disease about which we have little understanding and knowledge, it can often be misdiagnosed in clinical settings. Missense mutations in the POR gene were seen to be the most prevalent type of mutation causing PORD as compared to other mutations. This study aims to map the missense mutations, reported in PORD patients, on the 3D X-ray crystal structure of the POR enzyme, using a published human POR X-ray crystal structure (PDB: 5FA6). The objectives are to establish a potential correlation between the various POR mutations and their impact on the tertiary

POR Deficiency (PORD)
A number of gene mutations have been reported in the POR enzyme, resulting in the distortion of its 3D protein structure, ultimately causing POR to function abnormally. The mutation-driven changes in the 3D structure of the POR enzyme could lead to a medical condition known as cytochrome P450 oxidoreductase deficiency (PORD) (OMIM: 613571), which is an autosomal recessive genetic disorder and can also be categorized as a rare congenital adrenal hyperplasia (CAH) [3,8]. PORD has an impact on the regulation of steroid hormone production in the body; thus, this condition could affect the normal development of the skeletal system, the reproductive system, as well as metabolic pathways. In an early experiment, Peterson et al. (1985) [9] assessed a 46, XY, six-month-old infant characterized by having a female phenotype and abnormal genitals, with an unusual steroid profile, and it was assumed to be caused by a loss of function of both CYP17A1 and CYP21A2. This report led Miller et al. (1986) [10] to propose that there might be a defect in the POR enzyme, which provides electrons to both CYP17A1 and CYP21A2. In spite of that, the hypothesis was excluded as an embryonic lethality was discovered in POR knockout mice [11,12]. It was not until in 2004, when Flück et al. [3] confirmed that the first four individuals suffering from PORD have mutations in their POR gene. The author also described how three out of four patients developed abnormal genitals while the remaining 1 patient suffered from an abnormal steroid profile, a cystic ovary, and primary amenorrhea [3].
As PORD is a rare autosomal recessive disease about which we have little understanding and knowledge, it can often be misdiagnosed in clinical settings. Missense mutations in the POR gene were seen to be the most prevalent type of mutation causing PORD as compared to other mutations. This study aims to map the missense mutations, reported in PORD patients, on the 3D X-ray crystal structure of the POR enzyme, using a published human POR X-ray crystal structure (PDB: 5FA6). The objectives are to establish a potential correlation between the various POR mutations and their impact on the tertiary structure and function of the POR enzyme, as revealed by observing the clinical phenotypes reported in PORD patients [13][14][15][16].

Compilation of Reported POR Genetic Variants
We have identified a total of approximately 170 cases of PORD reported around the globe since its initial discovery in the year 2004 (Table 1). The extent of the POR enzyme impairment dictates the clinical phenotype, such as disorders of sex development (DSDs), irregular steroid hormone profiles, and skeletal malformations observed in PORD patients. Around 84.1% of PORD patients display ABS features and 70.6% of patients experienced ambiguous external genitalia (Table 1). Furthermore, the tabulated data indicated that a higher number of female individuals with 46, XX karyotype were seen to suffer more from PORD as compared to male, at a ratio of 1.3:1 (Table 1). In addition, a total of 32 reported POR genetic variants, caused by missense mutations in the POR gene, were identified and listed in Table 2, representing 25 disease-causing and 7 polymorphic variants. The identified 32 genetic variants were further mapped on the primary protein sequence ( Figure 4) and tertiary structure of the human POR enzyme ( Figure 5) using an in silico analysis approach to establish their location within the different cofactor binding domains of the POR enzyme (Table 2).

In Silico Analysis of POR Mutations
To identify the highly conserved amino acid residues in the POR genome across different species, a multiple sequence alignment of human and five POR ortholog proteins (rat, monkey, squirrel, lynx, and dog), sharing above 90% sequence identity, was performed ( Figure 6). The sequence analysis identified several highly conserved amino acid residues among the POR orthologs (Figure 6), highlighting the importance of these conserved residues in maintaining the stability of the POR 3D structure and function, as they were retained throughout evolution. The interactions between different amino acid residues within a protein sequence have a great impact on the overall stability of the protein [55], and any change in amino acid residue(s) due to the mutation(s) could drastically influence the structural integrity of the protein [56,57]. The identification of conserved residues within the POR orthologs further prompted us to investigate the location of these conserved amino acid residues on different domains of the POR enzyme in order to understand their potential role in keeping the functional and structural integrity of the POR enzyme.

In Silico Analysis of POR Mutations
To identify the highly conserved amino acid residues in the POR genome across different species, a multiple sequence alignment of human and five POR ortholog proteins (rat, monkey, squirrel, lynx, and dog), sharing above 90% sequence identity, was performed ( Figure 6). The sequence analysis identified several highly conserved amino acid residues among the POR orthologs (Figure 6), highlighting the importance of these conserved residues in maintaining the stability of the POR 3D structure and function, as they were retained throughout evolution. The interactions between different amino acid residues within a protein sequence have a great impact on the overall stability of the protein [55], and any change in amino acid residue(s) due to the mutation(s) could drastically influence the structural integrity of the protein [56,57]. The identification of conserved residues within the POR orthologs further prompted us to investigate the location of these conserved amino acid residues on different domains of the POR enzyme in order to understand their potential role in keeping the functional and structural integrity of the POR enzyme. Therefore, using in silico analysis, we have mapped the 32 POR missense mutant variants on the crystal structure of POR enzyme. The in silico analysis reveals the presence of 9 out of the 32 POR variants on the FMN binding domain (A115V, T142A, G144S, Q153R, G177R, Y181D, N185K, D210G, and P228L) ( Figure 5). One of the mutation in the FMN binding domain, i.e., the Y181Dinteracts with the FMN cofactor using a pi-pi bond interaction ( Figure 7A) between the benzene ring of the tyrosine group and the isoalloxazine rings of FMN. Therefore, the substitution of tyrosine to aspartic acid at position 181 would result in the destabilization of the FMN cofactor. Several studies have determined that the Y181D mutation leads to a significant loss of activity of cytochrome c and the CYP17A enzyme [1,58,59], leading to bone defects, i.e., midface hypoplasia and phalangeal malformations [13,15]. In contrast, the D210G mutation presents patients with brachydactyly and external genitalia such as micro-penis and hypospadias [14]. The aspartic acid (D210) forms a hydrogen bond with neighboring residues K179 and D218 with an atomic distance of 2.12 Å and 1.76 Å, respectively ( Figure 7B), and its substitution to Glycine residue at position 210 would weaken these interactions, resulting in the distortion of the protein structure. As D210G was discovered recently, no POR activity has yet been associated with it. Therefore, using in silico analysis, we have mapped the 32 POR missense mutant variants on the crystal structure of POR enzyme. The in silico analysis reveals the presence of 9 out of the 32 POR variants on the FMN binding domain (A115V, T142A, G144S, Q153R, G177R, Y181D, N185K, D210G, and P228L) ( Figure 5). One of the mutation in the FMN binding domain, i.e., the Y181Dinteracts with the FMN cofactor using a pi-pi bond interaction ( Figure 7A) between the benzene ring of the tyrosine group and the isoalloxazine rings of FMN. Therefore, the substitution of tyrosine to aspartic acid at position 181 would result in the destabilization of the FMN cofactor. Several studies have determined that the Y181D mutation leads to a significant loss of activity of cytochrome c and the CYP17A enzyme [1,58,59], leading to bone defects, i.e., midface hypoplasia and phalangeal malformations [13,15]. In contrast, the D210G mutation presents patients with brachydactyly and external genitalia such as micro-penis and hypospadias [14]. The aspartic acid (D210) forms a hydrogen bond with neighboring residues K179 and D218 with an atomic distance of 2.12 Å and 1.76 Å, respectively ( Figure 7B), and its substitution to Glycine residue at position 210 would weaken these interactions, resulting in the distortion of the protein structure. As D210G was discovered recently, no POR activity has yet been associated with it.
Similarly, the analysis of the NADPH binding domain also reveals the presence of nine POR variants (G539R, L565P, C569Y, L577R, Y578C, E580Q, Y607C, V608F and H628P) ( Figure 5). The mutations in this group, e.g., Y607C, interact with the NADPH cofactor within the NADPH binding domain using a hydrogen bonds and pi-pi interactions ( Figure 7E), and its replacement with cysteine at position 607 is reported to have a significant loss in 17,20 lyase and aromatase enzymes activities [59,60], resulting in the reported clinical phenotype of bone deformities and disordered sex developments in both male and female patients [13,27]. nine POR variants (G539R, L565P, C569Y, L577R, Y578C, E580Q, Y607C, V608F and H628P) ( Figure 5). The mutations in this group, e.g., Y607C, interact with the NADPH cofactor within the NADPH binding domain using a hydrogen bonds and pi-pi interactions ( Figure 7E), and its replacement with cysteine at position 607 is reported to have a significant loss in 17,20 lyase and aromatase enzymes activities [59,60], resulting in the reported clinical phenotype of bone deformities and disordered sex developments in both male and female patients [13,27].

Compilation of Reported Cases of POR Mutations
All mutation variants from the reported cases of PORD were listed and tabulated according to their mutated site, mutation residue, DNA change, location on the exon, as well as the domain affected by the mutation. As this study only focuses on missense

Compilation of Reported Cases of POR Mutations
All mutation variants from the reported cases of PORD were listed and tabulated according to their mutated site, mutation residue, DNA change, location on the exon, as well as the domain affected by the mutation. As this study only focuses on missense mutations reported in the POR gene, variants caused by other types of mutations such as frameshift, nonsense, insertion, deletion, duplication, and splice-site mutations are excluded.

Bioinformatics Analysis of POR Protein Sequences
The National Center for Biotechnology Information (NCBI) database was utilized in order to access the Homo Sapiens POR protein sequence (NCBI: NP_000932.3). Once the sequence was retrieved, the computer algorithm basic local alignment search tool (BLAST) (blast.ncbi.nlm.nih.gov; accessed on 25 June 2022) was used to analyze and select POR orthologs from other species that share a close percentage identity with the human POR protein sequences. Based on the BLAST result, species such as Pongo abelli (monkey), Tupaia Chinensis (squirrel), Lynx rufus (lynx), Canis lupus familiaris (dog), and Rattus norvegicus (rat) were selected as the orthologs sharing 99.56%, 94.69%, 94.10%, 93.36%, and 92.48% similarities with the human POR sequence, respectively. Lastly, to find and compare the conserved regions between the human POR sequence and the other selected POR orthologs, the Clustal Omega multiple sequence alignment program (EMBL-EBI, Cambridgeshire, UK) (www.ebi.ac.UK; accessed on 25 June 2022) was used for the alignment of amino acids. Hence, the protein sequences that were used include Homo Sapiens (NCBI: NP_000932.3), Pongo abelli (NCBI: XP_024105393), Lynx Rufus (NCBI: XP_046945282.1), Tupaia Chinensis (NCBI: XP_006155694.1), Rattus novergicus (NCBI: NP_113764.1), and Canis lupus familiaris (NCBI: NP_001171276.1).

In Silico Analysis of 3D X-ray Crystal Structure of POR Protein
A wild-type 3D X-ray crystal structure of POR protein (PDB: 5FA6) was retrieved from the Protein Data Bank (PDB). The Molsoft ICM browser tool (Molsoft LLC, San Diego, CA, USA), was used to analyze the 3D structure of the POR enzyme. In addition, several reported mutations in the PORD patients were analyzed and mapped on the wild-type 3D X-ray crystal structure, and an in-depth in silico structural analysis of the several POR mutations was performed.

Conclusions
PORD is a genetic steroid disorder with multiple clinical presentations, as observed in patients suffering with PORD. The severity of the PORD clinical phenotypes depends upon the mutated amino acid residue and its location within the three cofactor binding domains, i.e., the FAD, FMN and NADPH binding domains of the POR enzyme. The in silico mapping of missense mutations onto the human POR structure reveals that those amino acid residues that are in direct contact with the cofactor and are substituted for residues with smaller sidechains, e.g., R457 to H and Y181 to D, would result in the distortion of interactions with the cofactor molecule and will compromise the stability and folding of the enzyme. However, a substitution such as A287 to P would interfere with the integrity of a secondary structure element, as the substitution of alanine for proline would introduce a turn/bend in the beta sheet harboring the A287 residue. Patients with the A287P mutation experienced the most severe form of bone defects and sexual development disorders. On the other hand, for residues that display charged interactions within the local environment, such as the two polar residues Y607 and D210 when substituted with cysteine, a non-polar residue, there will be severe steric and electrostatic clashes in the local environment which will severely affect the folding, architecture and stability of the POR enzyme. In contrast, the missense mutations not directly interfering with the cofactor binding domains usually result in less structural destabilization and would present with less severe clinical phenotypes.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.